Although S deficiency has been reported in plants worldwide, the belowground biogeochemical cycling of S is not well known. The combined use of mineral fertiliser and manure is regarded as a suitable fertilisation strategy to maintain agricultural soil productivity. A long-term (1964–2018) field experiment was selected to determine how manure application affects soil gross S mineralisation and immobilisation, and plant-derived organic S, cysteine (Cys), and methionine (Met) biological decomposition by 35S, 14C, and 15N labelling. High organic manure application did not increase organic S content in the topsoil owing to the high mineralisation rate, but it increased the organic S content in subsoil where mineralisation rates were relatively lower. S immobilisation dominated gross S fluxes, and the highest SO42− immobilisation rates were recorded under medium manure application. Most plant-derived protein S was decomposed to SO42− after 15 min, and only approximately 30% was retained in the microbial biomass. Protein bioavailability may have a more dominant role in soil S mineralisation than soil S-containing amino acids, owing to its higher concentration. The immobilisation of SO42− was considerably slower than that of proteins and amino acids, which indicates that the microorganisms preferred organic S over inorganic S and that the use is driven by C demand rather than S demand. Moreover, the microbial community released SO42− and NH4+ after taking up Cys and Met, and the imbalance of elements between substrates and microbes played a dominant role in soil S cycling. This process was strongly regulated by the nature of the substrate; less SO42− was released from Met than from Cys. Among the three important processes for organic S decomposition—the uptake by microorganisms, SO42− release, and SO42− reuse—manure application had a greater effect on SO42− release during organic S decomposition. Overall, manure application increased S bioavailability owing to high S fluxes, and high- and low-molecular weight organic S could be rapidly decomposed to SO42−.